PROJECT SUMMARY/ ABSTRACT Many genes required for cancer formation also perform essential functions in normal cells. Elimination of such genes would, therefore, likely evoke unfortunate side effects on normal cells. Recent developments in the field of gene regulation suggest a possible solution to this problem. Transcription is typically activated by enhancers, regulatory DNA elements bearing a group of bound transcriptional activators. An enhancer can extend over many hundreds of base pairs, and is often positioned hundreds or even thousands of base pairs from its target gene. A given gene is typically driven by an enhancer in one cell type, and by a different enhancer (comprising different transcription factors, bound at a different DNA location) in other cell types. We and others have described enhancers of oncogenes in tumor cells that are not found in normal cell s. Were we to inactivate or eliminate such tumor-specific enhancers (while not damaging enhancer formation in their non- tumor counterparts), we would inhibit tumor cell growth without affecting growth or function of normal cells. Three recent developments have suggested a path to identifying and inactivating specific enhancers. First, we and others have developed assays that identify enhancers genome-wide in tumors and in normal cells. Second, recent developments in CRISPR technology have suggested a means by which we can simultaneously eliminate (destroy) multiple independent enhancers. Third, methods have been developed to grow organoids, groups of cells that more closely mimic cells (normal and tumor) in vivo than previously described procedures. Key personnel on this application, Dr. Scott Lowe at Memorial Sloan Kettering Cancer Center (MSKCC), has made seminal advances in modeling cancer, using organoids. Our co -PI, Dr. Michael Roehrl at MSKCC, is a recognized physician-scientist and pathologist with special expertise in colorectal cancers (CRCs). Additional key personnel, serving as contributors, such as Dr. Christina Leslie also at MSKCC, is an expert in computational biology and will assist us in data analysis. We will identify CRC-specific enhancers in CRC tumors and organoids derived therefrom. We will apply CRISPR technology to identify which of the genes activated by these enhancers, in pairs, are required for growth of CRC organoids. We will then use CRISPR tiling mutagenesis to identify enhancer sequences required for expression of these genes. From this knowledge, we will design pairs of guide RNAs that, by simultaneously attacking crucial enhancers, will specifically inhibit CRC organoid formation.